Project Summary The lack of accurate and reliable techniques for visualizing and quantifying the delivery and uptake (pharmacokinetics) of small molecules into the skin has been a major challenge in the development of both new and generic topical drugs. This is particularly true in the measurement of bioequivalence, where a generic drug candidate must be compared against a reference brand-label drug. Current methods, which rely on expensive, destructive, and bulk measurements do not provide the spatial or temporal resolution necessary to extract pharmacokinetic profiles to determine equivalence of drugs at the cellular level over time in human skin. New tools, which allow for direct, real-time quantification of pharmacokinetics non-invasively in humans are needed to drive innovation in the topical drug space. Pharmacokinetic Tomography (PKT) is a method of generated three-dimensional, real-time maps of various molecular species such as lipids, water, proteins and drugs in tissue using spectroscopic imaging techniques. These imaging techniques make use of the intrinsic molecular vibrations of drug molecules to track their uptake quantitatively without the need for labels or alterations to molecular structure. This proposal will develop new coherent Raman imaging tools to create a platform for quantifying the behavior of drugs in ex vivo human skin. This PKT-based platform will be able to measure the uptake of drugs such as antibiotics, kinase inhibitors, steroids, retinoids and NSAIDs in the skin with sub-cellular and sub-second time resolution to glean critical pharmacokinetic parameters needed for drug development and bioequivalence measurements. To compare PKT to an FDA-recognized standard, these PKT imaging will be carried out alongside dermal open flow microperfusion (dOFM), a skin fluid sampling technique that measures the concentration of drugs over time. The multimodal PKT platform, when completed, will be able to measure the bioequivalence of various drugs in situ in human subjects. This research program will specifically develop new imaging toolkits that combine advanced microscopy with state-of-the-art computer vision and machine learning analysis techniques to collect and extract pharmacokinetic data. Using a new type of versatile laser, we will create a coherent Raman technique ideal for detection of drug molecules which contain unique chemical structures. In order to broaden the applicability of the platform to a far wider range of potential drug molecules, we will develop a sparse sampling coherent Raman imaging approach that will quantify molecules by their unique combinations of molecular structures.